The Luteinizing Hormone Receptor (LHR): The LHR is expressed primarily in the gonads where it mediates LH signals that regulate ovarian and testicular function. The LHR gene transcription is regulated by complex and diverse networks, in which coordination and interactions between regulatory effectors are essential for silencing/activation of LHR expression. The proximal Sp1 site of the promoter recruits histone (H) deacetylases and the Sin3A corepressor complex that contributes to the silencing of LHR transcription. Site specific acetylation/methylation-induced phosphatase release serve as switch for Sp1 phosphorylation, recruitment of TFIIB and Pol II and transcriptional activation. Maximal derepression of the gene is dependent on DNA demethylation of the promoter, H3/H4 acetylation and HDAC/Sin3A release. Positive Cofactor 4 (PC4) has an important role in the formation/assembly of PIC in TSA-mediated LHR transcription (Liao & Dufau JBC 2011). It is recruited by Sp1 following TSA treatment and acts as its coactivator. However, PC4 does not participate in TSA release of phosphatases, Sp1 phosphorylation or release repressor/complexes. Although TFIIB recruitment is dependent on PC4 we have ruled out TFIIB as its direct target. However, TSA induced acetylation of a PC4 interacting proteins, was identified as Acetylated H3 by MS, and its presence in the complex in association to chromatin at the promoter was demonstrated by ChiP/reChiP. The role of these interactions on chromatin structure and their participation in the assembly of the PIC and transcriptional activation are under investigation.Immunoprecipitated flag-tagged PC4/H3-complexes in transfected MCF-7 cells analyzed by immunobloting using specific antibodies to acetylated H3 revealed Acetyl H3K4,K9,K14,K18,K23 pulled down by Flag Ab. To elucidate the physiological impact of PC4 on Sp1 directed transcription in gonads, we are generating a PC4-floxed mice to be bread with trangenic mice expressing tissue specific Cyp17 Cre or AMH Cre. Gonadotropin regulated Testicular RNA Helicase (GRTH/DDX25): GRTH is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig cells (LC) and meiotic germ cells. It is a multi-functional protein essential for the completion of spermatogenesis. Males lacking GRTH are sterile due to the absence of sperm resulting from failure of round spermatids to elongate. In addition, to its intrinsic RNA helicase activity, GRTH is a shuttling protein that exports specific mRNAs from the nucleus to cytoplasmic sites. Our studies have demonstrated the essential participation of the GRTH export/transport of mRNAs in the structural integrity of the Chromatoid Body (storage/processing of mRNAs) and their transit/association to actively translating polyribosomes where it may regulate translational initiation of genes. We have identified mRNAs which are associated with GRTH and regulated at polysomal sites of cell populations of the mouse testis. The reduction in mRNAs associated at polysomal sites in the differential studies (KO vs WT) not detected at total cellular level but in the cytoplasm with abolition of protein expression are reflective of the importance of the transport function of GRTH to relevant sites and underscore its impact in protein synthesis. GRTH is regulated by LH through androgen (A) at the transcriptional level in LCs (direct) and germ cells (presumably indirect) of the testis where its expression is both cell- and stage specific. This helicase displays a novel negative autocrine control of androgen production in LCs by preventing overstimulation of the LH-induced androgen pathway through enhanced degradation of StAR protein. Androgen (A)/Androgen Receptor (AR) regulates the expression of the GRTH gene in the Leydig cell via a short range chromosomal loop. A/AR signaling in LCs through its activation of GRTH transcription, participates in an autocrine regulation mechanism with a major impact on Leydig cell steroidogenic function. Our development of transgenic mice model carrying GRTH 5' flanking regions-GFP reporter provides a unique in vivo system that permits differential elucidation of regulatory regions in the GRTH gene that directs its expression (upstream) in germ cells (pachytene spermatocytes and round spermatids) and downstream in LCs and its regulation by A/AR in LC (directly) and indirectly in germ cells (1). Functional binding sites for germ cells specific transcription factor (GCNF) and its regulation by A/AR was identified in the distal region. In contrast, the proximal region directs basal GRTH expression and androgen-induced intracrine expression in Leydig cells through a functional ARE. This model permits to elucidate mechanism for androgen action in germ cells that would permit the identification of androgen regulated factors that control expression of a critical gene(s) require for GRTH expression in germ cells involved in the progress of spermatogenesis. This could lead to development of contraceptive strategies, that block sperm formation without impacting other aspects of androgen action. Prolactin receptor (PRLR): The PRLR is a member of the lactogen/cytokine receptor family which mediates the diverse cellular actions of Prolactin (PRL). PRL is a major factor in the proliferation and differentiation of breast epithelium and is essential for lactation. It has been also implicated in the development of breast cancer, tumoral growth and chemoresistance. hPRLR expression is controlled at the transcriptional level by multiple promoters (one generic, PIII, and five human specific) that were defined and characterized in our laboratory. Each promoter directs transcription/expression of a specific non-coding Exon 1 (E1-3, hEN1-hEN5), a common non-coding exon 2 and coding exons (E3-E11). The transcription of PRLR in breast cancer cells is directed by the preferentially utilized PIII that lacks an ERE. BRET studies revealed ERa constitutive homodimers. Complex formation of ERa dimer (non-DNA bound)with Sp1 and C/EBPb dimers bound to their sites at the promoter is required for basal (constitutive ERa homodimers)and E2-induced transcriptional activation/expression of the human PRLR gene. PRL in tumoral breast causes cell proliferation via activation of its cognate receptor. Exacerbation of PRL's actions resulting from increased receptor number can explain resistance to E2 inhibitors in breast cancer. Our studies in MCF7 cells reveal stimulation of PRLR promoter activity/mRNA/protein in cells by PRL in absence of E2 abolished by mutation of a GAS site, Stat5 siRNA, or ERa-antagonist. This indicates the participation of the ERa in PRLR transcription via PRL/PRLR/Stat5. PRL/PRLR induces phosphorylation of ERa through JAK2/PI3K/MAPK/ERK and JAK2/HER2 activated pathways. Increased recruitment of phosphoER-to Sp1 and C/EBPb bound at promoter sites is essential for PRL-induced PRLR transcription. Direct evidence is provided for local actions of PRL independent of E2 in the up-regulation of PRLR transcription/expression via a STAT5/ER activation-loop. The studies are of relevance in refractory states to aromatase inhibitors where cancer progression could be fueled by endogenous PRL. Therapies that inhibit the function of PRL or PRLR combined with inhibitors targeting the various signal transduction pathways could improve reversing resistance in breast cancer. Further targeting ERa and PRLR could eliminate constitutive activation of ER and PRLR by endogenous PRL and circunvent resistance(3). Other studies demonstrated essential role of D1 domain of the PRLR short-form structure and its inhibitory action on PRL-induced long form-mediated function. Changes in PRLR structure and dimerization affinity are triggered by single mutations in D1 providing avenues for breast cancer treatment(2)